In vitro replication of plasmids containing human ribosomal gene sequences: origin localization and dependence on an aprotinin-binding cytosolic protein.

We previously investigated the role of an aprotinin-binding protein (ADR) in the initiation of DNA replication in isolated quiescent nuclei. In the present study, we have used a cell-free DNA replication system to test the ability of plasmid vectors which contain sequences from the human ribosomal RNA gene to serve as replicative templates in vitro when exposed to ADR-containing preparations. Significant dTTP incorporation was seen using DNA from either a 7-kb sequence in the 5' spacer region (CHE) or a 7-kb sequence which begins near the end of the 28S coding region and extends into the 3' spacer region (ADBB), while sequences from other regions of the rRNA gene mediated little or no dTTP incorporation. The characteristics of plasmid-directed dTTP incorporation indicate that most incorporation is due to DNA replication and not repair or damage-initiated processes. To conclusively demonstrate origin-dependent replication in the plasmid system and to further map replication origins, an approach was developed using ddGTP to restrict the length of daughter strands followed by hybridization of these replication products to restriction fragments spanning the putative origin region. This approach allowed us to identify replication origin activity apart from parent strand repair or synthesis initiated at random damaged sites. One of the origins was localized to a 1375-bp fragment within the 5' spacer region, and this fragment contains sequences homologous to those found in other replication origins.